Identification of salmonella typhimurium virulence gene Ict and preparation and application of attenuated vaccine candidate strain of salmonella typhimurium virulence gene Ict

Abstract

The invention discloses identification of a salmonella typhimurium virulence gene Ict and preparation and application of an attenuated vaccine candidate strain of the salmonella typhimurium virulence gene Ict. According to the invention, a virulent salmonella typhimurium SL1344 strain is selected as a parent strain, and a key virulence gene Ict for degrading and metabolizing itaconic acid in the virulent salmonella typhimurium SL1344 strain is knocked out to construct a salmonella typhimurium SL1344 attenuated vaccine candidate strain. A mouse animal toxicity experiment, an immune protection experiment, a safety experiment and the like prove that the vaccine candidate strain SL1344-delta Ict deletion strain not only has obviously reduced pathogenicity to animals, but also provides good immunogenicity and immune protection. The attenuated salmonella typhimurium strain disclosed by the invention can be used for attenuating the virulence salmonella typhimurium strain, can be widely applied to prevention and control of salmonella, and lays a foundation for developing attenuated live vaccines and live vector vaccines of salmonella.

Description

Technical Field

[0001] The invention relates to the identification of the virulence gene Ict of Salmonella typhimurium and the preparation and application of attenuated vaccine candidate strains thereof, and belongs to the application field of the Ict gene. Background Art

[0002] Salmonella is an important zoonotic pathogen that is mainly transmitted through water and food, and can cause food poisoning, acute gastric typhoid, sepsis and other diseases. There are many serotypes of Salmonella, and Salmonella typhimurium, as the dominant serotype of Salmonella infection, can infect a variety of poultry and livestock, and can also cause human food poisoning and a variety of foodborne diseases. Salmonella has caused huge economic losses to the country and even the world, and has seriously threatened people's health. Therefore, controlling Salmonella infection is of great significance to protecting people's public health safety.

[0003] Itaconic acid, also known as methylene succinic acid, is a white crystalline, highly polar α,β-unsaturated dicarboxylic acid (C5H6O4) with double bonds and two carboxyl groups. It is similar in structure to phosphoenolpyruvate, succinic acid, maleic acid and fumaric acid. This similarity has laid a solid theoretical foundation for the research of itaconic acid in the field of antibacterial and immunomodulatory. The survival and reproduction of pathogens in eukaryotic hosts are highly dependent on two major metabolic cycles: the tricarboxylic acid cycle (TCA cycle) and the glyoxylate cycle. Itaconic acid is an important immunomodulatory small molecule synthesized by mitochondrial aconitate decarboxylase in macrophages under the stimulation of pathogens. Itaconic acid has a unique antibacterial effect. It can inhibit the activity of succinate dehydrogenase, prevent succinate from being oxidized to fumaric acid, and then inhibit the TCA cycle. At the same time, it is also an effective inhibitor of isocitrate lyase, inhibiting the glyoxylate cycle. However, some special pathogens (Yersinia pestis, Pseudomonas aeruginosa and Mycobacterium tuberculosis) can degrade and metabolize itaconate. The relevant metabolic pathways of itaconate metabolism in pathogens have been reported in the literature. The genes Ict (itaconyl-CoA transferase), Ich (itaconyl-CoA hydratase) and Ccl (S-citrate-CoA lyase) encoded by them catalyze the conversion of itaconate into acetyl-CoA and pyruvate, thereby making the pathogens tolerant to itaconate and survive in macrophages. In the early stage, through literature survey and bioinformatics analysis, it was found that the gene Ict related to itaconate degradation and metabolism is widely present in Salmonella, but the physiological and biochemical functions of itaconate metabolism gene Ict (Genebank: NC_016810.1) in Salmonella pathogens including Salmonella typhimurium are not clear.

[0004] At present, the main way to control Salmonella infection in China is antibiotics. However, the excessive use of antibiotics has caused the emergence of a large number of multidrug-resistant Salmonella. There are many beneficial bacteria in the body. The abuse of antibiotics will kill some beneficial bacteria while killing Salmonella, leading to intestinal flora imbalance, which will cause serious bacterial infections. As the problem of antibiotic resistance becomes increasingly serious, it is necessary to develop new and efficient vaccines. Vaccines mainly include inactivated vaccines and live attenuated vaccines. Live attenuated vaccines will not cause the occurrence of diseases when injected into the body, but pathogens can trigger the body's immune response, which plays a role in obtaining long-term or lifelong protection. Compared with inactivated vaccines, this type of vaccine has strong immunity and a long duration of action. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide the identification of the virulence gene Ict of Salmonella typhimurium and the preparation and application of the candidate strains of its attenuated vaccine.

[0006] Technical solution: To solve the above technical problems, the present invention provides the use of an Ict gene or a homologous gene thereof having a nucleotide sequence as shown in SEQ ID NO.1 in regulating the virulence of Salmonella.

[0007] The present invention also provides the use of the protein encoded by the Ict gene or its homologous gene in regulating the virulence of Salmonella.

[0008] The Ict gene is a gene identified from Salmonella typhimurium and has itaconic acid metabolism toxicity.

[0009] The present invention also provides the use of the Ict gene or its homologous gene with a nucleotide sequence as shown in SEQ ID NO.1 in regulating sensitivity to itaconic acid.

[0010] The present invention also provides the use of the protein encoded by the Ict gene or its homologous gene whose nucleotide sequence is shown in SEQ ID NO.1 in regulating the sensitivity of Salmonella to itaconic acid.

[0011] The present invention also provides an Ict gene-deficient strain, which is obtained by knocking out the Ict gene or its homologous gene of a wild-type Salmonella strain; the nucleotide sequence of the Ict gene is shown in SEQ ID NO. 1. The present invention uses 6-week-old BABL / C mice as a model to compare the difference in virulence between the gene-deficient mutant strain and the wild-type strain. The results of the lethality test show that the SL1344-ΔIct mutant strain has significantly lower virulence than the wild-type SL1344, and is a potential candidate live attenuated Salmonella typhimurium vaccine.

[0012] The present invention also provides a method for constructing the Ict gene deletion strain, comprising the following steps:

[0013] (1) Using the genomic DNA of Salmonella typhimurium as a template, PCR amplified the upstream and downstream homologous arms of the Ict gene, Ict-up and Ict-down fragments, and gel-recovered the Ict-up and Ict-down fragments; homologously recombined with the linear vector, and transformed into Escherichia coli to obtain the donor bacteria;

[0014] (2) The donor bacteria in step (1) are subjected to conjugation transfer and homologous single exchange recombination with Salmonella typhimurium, and the homologous double exchange is selected using chloramphenicol resistance and sucrose sensitivity to obtain the deletion strain.

[0015] Wherein, the nucleotide sequences of the primers for PCR amplification in step (1) are shown as SEQ ID NOs. 12 to 15.

[0016] Wherein, in step (1), Ict-up-F / R and Ict-down-F / R are used as primers, and the up and down sequences of the Ict gene are amplified by a high-fidelity enzyme.

[0017] Wherein, the nucleotide sequences of the primers for PCR amplification in the homologous double exchange in step (2) are shown in SEQ ID NOs. 18 to 19.

[0018] The present invention also provides a method for improving the sensitivity of Salmonella to itaconic acid, which is achieved by knocking out the Ict gene of Salmonella or its homologous gene; the nucleotide sequence of the Ict gene is shown in SEQ ID NO.1.

[0019] The present invention also provides a method for reducing the virulence of Salmonella, which is achieved by knocking out the Ict gene of Salmonella or its homologous gene; the nucleotide sequence of the Ict gene is shown in SEQ ID NO.1.

[0020] The present invention also provides an Ict gene or a homologous gene thereof with a nucleotide sequence as shown in SEQ ID NO.1, a protein encoded by the Ict gene or a homologous gene thereof, and an application of the deletion strain in preparing a vaccine for preventing Salmonella typhimurium.

[0021] The invention also provides a vaccine for preventing Salmonella, which contains the deleted strain.

[0022] Wherein, the Salmonella includes one or more of Salmonella typhimurium, Salmonella enteritidis, Salmonella infantis, Salmonella London or Salmonella Newport.

[0023] The invention firstly identifies the itaconic acid metabolism virulence gene Ict in Salmonella typhimurium SL1344, performs physiological and biochemical characterization on it and provides a Salmonella typhimurium attenuated vaccine candidate strain SL1344-ΔIct, which is obtained by knocking out the Ict gene of Salmonella typhimurium SL1344. The Salmonella typhimurium SL1344 is purchased from China Food and Drug Inspection Institutes.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention selects the highly virulent Salmonella typhimurium SL1344 strain as the parent strain, knocks out the key virulence gene Ict for degrading and metabolizing itaconic acid, and constructs a candidate strain of attenuated vaccine of Salmonella typhimurium SL1344; 2. Through virulence experiments on mice, immune protection experiments and safety experiments, it is confirmed that the vaccine candidate strain SL1344-ΔIct deletion strain not only significantly reduces the pathogenicity to animals, but also provides good immunogenicity and immune protection; 3. The present invention achieves attenuation of highly virulent Salmonella typhimurium strains, which can be widely used in the prevention and control of Salmonella, and lays the foundation for the development of attenuated live vaccines and live vector vaccines for Salmonella; 4. The highly conserved virulence gene Ict for degrading itaconic acid is commonly found in the genomes of Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Infantis, Salmonella London and Salmonella Newport. Therefore, the "preparation and application of the itaconate virulence gene Ict of Salmonella typhimurium in attenuated vaccine candidate strains" proposed in the present invention can be widely applicable to the preparation and application of the itaconate virulence gene Ict in attenuated vaccine candidate strains of Salmonella Enteritidis, Salmonella Infantis, Salmonella London, and Salmonella Newport. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The relative expression of Ict was detected by RT-qPCR;

[0026] Figure 2 PCR amplification of the target gene Ict;

[0027] Figure 3 This is the SDS-PAGE gel image of the target protein Ict;

[0028] Figure 4 For the analysis of the catalytic activity of Ict protein by liquid chromatography-mass spectrometry: Figure 4 A is the liquid chromatography UV peak of the reaction between acetyl-CoA and itaconic acid and Ict; Figure 4 B is the mass spectrum peak of the reaction between acetyl-CoA and itaconic acid and Ict;

[0029] Figure 5 PCR diagram for constructing the deletion strain SL1344-ΔIct: Figure 5A is the PCR amplification diagram of the target gene Ict-up and Ict-down; Figure 5 B is the double restriction enzyme digestion verification of plasmid pDM4; Figure 5 C is the target fragment and the vector connection transformation identification; Figure 5 D is PCR verification of single-swap strains; Figure 5 E is the external primer verification of the mutant strain SL1344-ΔIct screened on sucrose plate; Figure 5 F is the internal and external primer verification of the mutant strain SL1344-ΔIct; Figure 5 G is the verification of wild strain SL1344;

[0030] Figure 6 A is the growth curve of wild-type Salmonella typhimurium strain SL1344 and SL1344-ΔIct deletion strain in LB medium; Figure 6 B is the results of the sensitivity test of wild-type Salmonella typhimurium strain SL1344 and SL1344-ΔIct deletion strain to itaconic acid;

[0031] Figure 7 LD of wild-type Salmonella typhimurium strain SL1344 (A) and SL1344-ΔIct deletion strain (B) 50 ;

[0032] Figure 8 Results of the mouse immune protection experiment of the SL1344-ΔIct deletion strain: Figure 8 A is a schematic diagram of the mouse immune protection experiment; Figure 8 B is the result of mouse immune protection experiment;

[0033] Fig. 9 is the clearance rate of the SL1344-ΔIct deletion strain in the liver;

[0034] Fig.10 is the clearance rate of the SL1344-ΔIct deletion strain in the spleen;

[0035] Fig.11 is the clearance rate of the SL1344-ΔIct deletion strain in the cecum;

[0036] Fig.12 is the clearance rate of the SL1344-ΔIct deletion strain in the ileum;

[0037] Fig.13 is the distribution rate of SL1344-Ict gene in 9 common serotypes of Salmonella. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0039] Example 1 Identification and Physiological and Biochemical Characterization of the Itaconic Acid Degrading Gene Ict of Salmonella Typhimurium

[0040] 1. Characterization of the physiological function of the itaconate degradation gene Ict in Salmonella typhimurium at the transcriptional level

[0041] In order to study the physiological function of itaconic acid metabolism gene Ict in Salmonella typhimurium, Salmonella typhimurium SL1344 was cultured in M9 medium and M9 medium + 20mM itaconic acid, respectively. The relative expression level of Ict was detected by qRT-PCR after 18 hours of culture and compared with that of the M9 medium group without itaconic acid.

[0042] 1.1 Extraction of total bacterial RNA

[0043] Salmonella typhimurium SL1344 (purchased from China Food and Drug Inspection Institute) was inoculated into 5 mL of M9 medium (Na2HPO4 6.8 g / L, KH2PO4 3 g / L, NaCl 0.5 g / L, NH4Cl 1 g / L) and M9 medium + 20 mM itaconic acid, respectively, and cultured at 37°C for 18 h. The next day, all the bacterial liquid was centrifuged at 12000 rpm for 2 min, and the supernatant was discarded. The total RNA was extracted from the sample according to the instructions of the bacterial total RNA extraction kit (TIANGEN), and the resulting product was stored in a -70°C refrigerator.

[0044] 1.2 Reverse transcription of RNA samples

[0045] The reverse transcription reaction of bacterial RNA was performed using the reverse transcription kit of Vazyme according to the following reaction conditions. The genomic DNA removal and reverse transcription reaction system are shown in Table 1:

[0046] Table 1. Removal of gDNA and RNA reverse transcription system

[0047]

[0048] The reaction conditions are as follows: Step 1: prepare the mixture of reaction 1 in a centrifuge tube, mix well, and incubate at 42°C for 2 minutes; Step 2: add 5×HiscriptⅢqRT Super Mix to the tube containing the mixture of the previous step and mix well. The system is incubated in a water bath at 37°C for 15 minutes for reverse transcription, and incubated in a metal bath at 85°C for 5 seconds to inactivate the reverse transcriptase in the system. RNase-free water is added as needed for dilution and stored at -70°C for later use.

[0049] 1.3 Fluorescence quantitative PCR

[0050] The reaction system in Table 2 was prepared according to the instructions of AceQ Universal SYBR qPCR Master Mix (Vazyme):

[0051] Table 2 Fluorescence quantitative PCR reaction system

[0052]

[0053] The fluorescence quantitative PCR reaction program was as follows: 95°C for 10 min, 95°C for 10 s, and 60°C for 1 min, for a total of 40 cycles.

[0054] Among them, the primers required for fluorescent quantitative PCR were designed according to the full genome sequence of the standard strain of Salmonella typhimurium SL1344 published on GenBank (NC_016810.1) and synthesized by Qingke Biotechnology Co., Ltd. The primer sequences are shown in Table 3:

[0055] Table 3 qRT-PCR detection of Ict gene expression

[0056]

[0057] Among them, the reference gene gyrA is numbered A0A718RXE8 in Uniprot.

[0058] like Figure 1 As shown, according to the above conditions, the whole genome sequence of the standard strain of Salmonella typhimurium SL1344 was used as a template for fluorescence quantitative PCR. The RT-qPCR experiment found that compared with the M9 medium group (Glucose) without itaconic acid, the itaconic acid metabolism gene Ict was significantly induced in the Salmonella typhimurium SL1344 (Itaconate) cultured with 20mM itaconic acid. Therefore, it was confirmed from the transcriptional level that Ict plays an important physiological function in itaconic acid metabolism.

[0059] 2. Characterization of the biochemical function of the itaconate degradation gene Ict in Salmonella typhimurium

[0060] 2.1 Extraction of genomic DNA from Salmonella typhimurium SL1344

[0061] A single colony of Salmonella typhimurium SL1344 was picked from the LB solid plate, inoculated into an antibiotic-free LB liquid medium, and cultured at 37°C with a shaker at 180 rpm for 12 h. 2-4 ml of the bacterial solution was used to extract the genomic DNA of Salmonella typhimurium SL1344 according to the instructions of the bacterial genome extraction kit (TIANGEN), and the product concentration was determined using a Nanodrop ultra-micro spectrophotometer, and stored in a -20°C refrigerator for later use.

[0062] 2.2 Amplification and recovery of Ict gene fragments

[0063] The primers required for the construction of the prokaryotic expression strain were designed based on the full genome sequence of the Salmonella typhimurium standard strain SL1344 published on GenBank, as shown in Table 4, and synthesized by Qingke Biotechnology Co., Ltd. The primer sequences are shown in the table:

[0064] Table 4 Primer sequences used to construct prokaryotic expression strain (pET28a-Ict)

[0065]

[0066] Using the extracted Salmonella typhimurium SL1344 genomic DNA as a template, the upstream primer Ict-F and the downstream primer Ict-R were used to amplify the Ict fragment, and the reaction system shown in Table 5 was prepared:

[0067] Table 5 PCR amplification system

[0068]

[0069] The PCR reaction conditions were as follows: pre-denaturation at 98°C for 15 s; denaturation at 98°C for 10 s, annealing at 60°C for 10 s, extension at 72°C for 10 s, for a total of 30 cycles, complete extension at 72°C for 2 min, and storage at 4°C.

[0070] The PCR products were identified by electrophoresis using 1% agarose gel. Figure 2 , accurately cut the target band and perform gel recovery on the target fragment according to the instructions of the agarose gel DNA recovery kit (TIANGEN), and the obtained product is stored in a -20°C refrigerator for future use. Among them, the sequence of Ict is shown in SEQ ID NO.1:

[0071] atgatgaatgttaatgcagtgtatgccgaaaaatgcgtgacgcctgacgaggcggttattattattacctccggcagtcacctctctatgg

[0072] gaatgtttgcagcagagcctccagctttgctgaatgcgctggcaaagcgagcgaaaaggggagaaatcaacgatctgcgggtttactg

[0073] ttatgaaacagccagcattgccgggaacactattttccgttatgagctaagtgactatattcatctctacagtatgtttattaccggtattgaa

[0074] cgggctttaatacgtcaggggattgagagttccgggcgaaaaatagttaactatgtaccttcaaattttcatcaggcgacgcgattactag

[0075] ctgatgatattggtattgatacctttattcacaccgtgtcccctatggataagtatggctattttaattttggaactggaaacgattattccaccc

[0076] ggattgcgaggacagctaaaaagcttattgtcgaagttaacaaatatatgccgcgcgttcatggagaaggtgctgcaatccatatttcag

[0077] aaattgacgccattgttgaaaaccatgtccctttaattgaactaccaatacgtactgccgtagccgaagatattgctatcagtcagattatcg

[0078] cttctcttgtgccagatggcgcttgtctccagatgggggtcggcgcattaccggagctcatctgtaatgcgctcaaggaacataatgatct

[0079] gggggtccatacagaggcgctgaatccggggttggtaagtttgattcaacaaggcgtcgttaccaatcagcgtaaaaatatcgaccgg

[0080] ggaatgtctgtatttacttttgctatgggacaaaaggatatgtatgactatctcaacgacaacccctctttttttagtcggcctgtcgattatgtt

[0081] aacgatccggggatcattgcgcaaaatgagaatgttgtctccattaacgctaccttacaaatcgatctcactggtgcctgtaactctgaata

[0082] tttgcttggacaccagtacagcgcttcaggagggcaattggattttgtcagaggcgcatatgcatctaaaggaggtcgttccattattacg

[0083] acccgctcaacggccgcgaatgatacgatttctcgtattgttccccgccttgaggggcctgtaacaaccccacgaacggatacgcactg

[0084] gatagtcactgaatttggctccgtaaacctgaaggggctctcttcaaccgagcgtgcgctcagcatcattgaattggcacatcccaatttt

[0085] cggcagcaacttagagtagatgcaaaaaaaatgcatctgatttaa

[0086] The Ict gene is numbered A0A0H3NLY8 in Uniprot.

[0087] 2.3 Plasmid pET28a digestion and homologous recombination transformation of Ict

[0088] 37°C, 4h digestion (SspI) pET28a plasmid, the digestion system is as follows:

[0089] Table 6 Enzyme digestion system

[0090]

[0091] After the enzyme digestion, nucleic acid electrophoresis was performed to verify whether pET28a was cut. The digested pET28a was purified using a gel recovery kit. The purified product was ligated at 50°C using a ligase. The ligation system was as follows:

[0092] Table 7 Homologous recombination system

[0093]

[0094] The ligated product was transferred into DH5α competent cells. After a single colony grew on the plate, a single clone was picked out and inoculated into 5 ml of LB liquid culture medium containing Kan (50 μg / ml). The culture was carried out at 180 rpm and 37°C for 12-16 h, and the recombinant plasmid was extracted. The extracted plasmid was sent to the company for sequencing.

[0095] 2.5 Expression of Ict protein

[0096] a. Transformation: The recombinant plasmid after successful sequencing was transferred into BL21 (DE3) competent cells. After a single colony grew on the plate, a single clone was picked and inoculated into 10 ml of LB liquid culture medium containing Kan (50 μg / ml) and cultured at 200 rpm and 37°C overnight.

[0097] b. Expansion culture: Take 3 ml of the culture medium obtained in step a and transfer it into 300 ml of LB liquid medium containing Kan (50 μg / ml), and culture at 37°C 200 rpm for 4-6 hours until OD 600 It is 0.6-0.8.

[0098] c. IPTG induced expression: When the culture medium OD 600 When the pH reached 0.6-0.8, IPTG was added to make the final concentration 0.4 mM and induced overnight at 18°C ​​and 200 rpm.

[0099] 2.6 Purification of Ict protein

[0100] The recombinant plasmids constructed in this experiment were all loaded into the vector with MBP tag

[0101] a. Bacteria collection: centrifuge the bacterial solution at 6000 rpm for 3 min, collect the bacteria and resuspend them in 20 ml of bacterial resuspension solution (20 mM Tris-HCl pH 7.5, 1 mM PMSF, 0.03% Triton X-100, 0.2 M KCl).

[0102] b. Disruption: Disrupt the resuspended bacteria in an ice-water bath. Set the ultrasonic cell disruptor to 25 Hz and disrupt for 3 seconds and then stop for 2 seconds for a total of 30 minutes.

[0103] c. Centrifugation: After crushing, centrifuge at 8000 rpm, 4°C for 10 min, collect the supernatant, and filter it with a 0.45 μm filter.

[0104] d. Purification: Purify the protein in the following order:

[0105] (1) Wash the gravity column with 10 ml of ddH2O;

[0106] (2) Equilibrate the gravity column with 10 ml of buffer A (20 mM Tris-HCl pH 7.5, 5 mM β-mercaptoethanol, and 0.2 M KCl);

[0107] (3) Loading the sample, passing the supernatant collected in step c through the gravity column;

[0108] (4) Elute the impurities with 10 ml of buffer A;

[0109] (5) The gravity column was eluted with 10 ml of buffer B (20 mM Tris-HCl pH 7.5, 5 mM β-mercaptoethanol, 0.2 M KCl and 1 mM maltose), the flow-through was collected, and the product concentration was measured using a Nanodrop ultra-micro spectrophotometer.

[0110] e.SDS-PAGE detection: Detect the flow-through obtained in step d and identify the target protein, such as Figure 3 Shown are 1, 2, and 4 μg of the purified target protein.

[0111] 2.7 Liquid chromatography-mass spectrometry

[0112] 300 μL reaction buffer including 20 mM Tris-HCl (pH 7.5), 5 mM itaconic acid, 0.5 mM acetyl-CoA and 5 μM Ict protein was incubated at room temperature for 1 hour. No Ict protein was added to the control group. At the end of the experiment, the reaction mixture was mixed with chromatography-grade methanol (1:1, v / v) and centrifuged at 15,000 g for 5 min. The supernatant was taken and filtered with a 0.22 μm filter.

[0113] The liquid chromatography column was ZORBAX SB-C18 (250×4.60mm, 5μm, product number 880975-902), the flow rate was 0.5mL / min at room temperature, and the column temperature was 24°C. 10mM ammonium formate (solvent A) and acetonitrile (solvent B) were used as mobile phases, and the gradients used were 0-1min, 2% B; 1-11min, 2→20% B; 11-13min, 20% B; 13-13.1min, 20→2% B; 13.1-23min, 2% B, the injection volume was 5μl, and UV detection was 260nm. Mass spectrometry detection was performed in positive ion mode.

[0114] The results showed that in positive ion mode, acetyl-CoA (m / z = 810.1337) and itaconic acid reacted with Ict, resulting in a decrease in the acetyl-CoA peak and the appearance of new peaks of itaconyl-CoA (m / z = 880.1374) and CoA (m / z = 768.1226) ( Figure 4 ), thereby identifying the function of the gene Ict that degrades itaconate at the biochemical level.

[0115] Example 2 Preparation of attenuated Salmonella typhimurium vaccine candidate strain SL1344-ΔIct

[0116] 1. Construction of suicide plasmid

[0117] 1.1 Primer design and amplification of Ict-up and Ict-down fragments of upstream and downstream homology arms

[0118] According to the genome sequence of Salmonella typhimurium published in GenBank, specific primers were designed using Snapgene, as shown in Table 9. The PCR system for amplifying Ict using the genome DNA of Salmonella typhimurium SL1344 as a template is shown in Table 8:

[0119] Table 8 Ict-up / down gene PCR amplification system

[0120]

[0121] The reaction conditions were as follows: pre-denaturation at 98°C for 15 s; denaturation at 98°C for 10 s, annealing at 60°C for 10 s, extension at 72°C for 10 s, for a total of 30 cycles, extension at 72°C for 2 min, and storage at 4°C.

[0122] Table 9 Ict-up / down gene primer sequences

[0123]

[0124] After the reaction, 1% agarose gel electrophoresis was used to detect the Figure 5 As shown in A, the kit recovers and purifies the PCR product, and the recovery operation is performed according to the TIANGEN instructions. Wherein, M: marker DL2000; Lane 1: PCR amplification diagram of the target gene Ict-up; Lane 2: PCR amplification diagram of the target gene Ict-down.

[0125] 1.2 Double restriction digestion of pDM4 vector

[0126] Table 10pDM4 vector double restriction enzyme digestion system

[0127]

[0128]

[0129] The enzyme digestion was carried out in a 37℃ water bath for 4h, and the digestion products were subjected to 1% agarose gel electrophoresis. Figure 5 As shown in B, the kit is recovered and purified. Among them, M: marker DL15000; Lane 1: the fragment of plasmid pDM4 after double digestion with SalI and SacI.

[0130] 1.3 DNA fragments and pDM4 vector ligation

[0131] Table 11 Homologous recombination system

[0132]

[0133] The recombinant product pDM4-ΔIct (ΔIct is the spliced ​​fragment of Ict-up and Ict-down) was transformed into E. coliχ7213 in a 50°C metal bath for 1 h, and positive clones were screened on LB plates containing chloramphenicol (25 μg / mL) and DAP (50 μg / mL). The universal outer primers of pDM4 (SEQ ID NO.16-17) were used for verification. Figure 5 C, verify that the sequencing is correct and it is the conjugation donor bacteria. Among them, M: DL2000 marker; Lane 1-2: PCR verification using primers pDM4-SalI-up-F / pDM4-SacI-down-R (PCR verification system is shown in Table 12).

[0134] Table 12 PCR verification system

[0135]

[0136] The reaction conditions were as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 2 min, for a total of 30 cycles, extension at 72°C for 10 min, and storage at 4°C.

[0137] 2. Bacterial conjugation transfer

[0138] The donor bacteria E. coli χ7213 containing the recombinant plasmid was cultured in LB liquid supplemented with chloramphenicol (25 μg / mL) and DAP (50 μg / mL) until OD 600 is 0.6, and the recipient bacteria SL1344 are cultured in LB liquid to the same OD value. Take 1 mL each of the donor bacteria and the recipient bacteria, wash them twice with PBS, mix them, keep 100 μL of the resuspended bacteria, and drop them onto a 0.22 μm hydrophilic filter membrane on an LB plate containing DAP (50 μg / mL), and place the plate upright in a 37°C incubator for overnight culture. Blow down the bacterial moss with sterile PBS, take 100 μL on an LB plate supplemented with chloramphenicol (25 μg / mL), and culture at 37°C overnight. Use two pairs of primers, pDM4-SalI-up-F / Ict-out-F and pDM4-SacI-down-R / Ict-out-F, for PCR identification (the verification system is shown in Tables 13 and 14). If there is a pair that can amplify the target band, it is correct, and a single exchange strain is obtained, such as Figure 5 D. Among them, M: DL5000 marker; Lane 1-2: single exchange followed by PCR verification using primers Ict-Out-F / pDM4-SalI-up-F and Ict-Out-F / pDM4-SacI-down-R.

[0139] Table 13 PCR verification system 1

[0140]

[0141] Table 14 PCR verification system 2

[0142]

[0143] The reaction conditions were as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 3 min, for a total of 30 cycles, extension at 72°C for 10 min, and storage at 4°C.

[0144] 3. Screening of gene deletion strains

[0145] The single exchange strain was inoculated into LB liquid medium supplemented with chloramphenicol (25 μg / mL) for 4 h, expanded in LB liquid medium containing 15% sucrose at a ratio of 1:100 for 12-16 h, and then the bacterial liquid was dipped into LB solid medium containing 15% sucrose and cultured at 37°C for 12-24 h. Positive clones were selected to prepare templates, and PCR amplification was performed on them using Ict-out-F / R (Table 15) primers (the amplification system is shown in Table 16). Ict-out-F / R amplified wild strains as long bands and amplified deletion strains as short bands. The obtained deletion strains were inoculated on LB (Cm+) plates and XLT4 plates in three zones, cultured overnight, and single colonies (2-3) that only grew on XLT4 plates but not on Cm+ plates were selected to prepare templates. Ict-out-F / R primers and Ict-in-F / R primers were used for amplification (the amplification system is shown in Tables 16 and 17), respectively, compared with wild strains, run gel verification, and send the correct deletion strain PCR product for sequencing (sequencing by Qingke Biotechnology Co., Ltd.). The results are as follows Figure 5 E-5G. Among them, Figure 5 M in E: DL5000 marker; Lane 1: SL1344-ΔIct was verified using primers Ict-out-F / R; Figure 5 M in F: DL5000 marker; Lane 1: SL1344-ΔIct was verified using external primers Ict-out-F / R; Lane 2: SL1344-ΔIct was verified using internal primers Ict-in-F / R; Figure 5 M in G: DL5000 marker; Lane 1: The wild strain SL1344 was verified using primers Ict-out-F / R; Lane 2: The wild strain SL1344 was verified using primers Ict-in-F / R.

[0146] Table 15 Screening primers for deletion strains

[0147]

[0148] Table 16 PCR verification system 1

[0149]

[0150] Table 17 PCR verification system 2

[0151]

[0152] The reaction conditions were as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 2 min, for a total of 30 cycles, extension at 72°C for 10 min, and storage at 4°C.

[0153] Example 3: Biological analysis of the attenuated Salmonella typhimurium vaccine candidate strain SL1344-ΔIct

[0154] Source of SL1344: A strain of Salmonella Typhimurium purchased from the China Food and Drug Inspection Institutes.

[0155] 1. Growth curves of wild-type Salmonella typhimurium strain SL1344 and SL1344-ΔIct deletion strain in LB medium

[0156] The bacterial solution OD 600 The SL1344-ΔIct deletion strain and the wild-type SL1344 strain with a concentration of 1 were transferred to LB liquid medium and cultured in a shaking incubator at 37°C. The OD was measured every hour. 600 The growth curve was drawn based on the recorded data. The results showed that there was no significant difference in the in vitro growth rate between the wild-type strain and the deletion strain ( Figure 6 A).

[0157] 2. Results of the sensitivity experiment of wild-type Salmonella typhimurium strain SL1344 and SL1344-ΔIct deletion strain to itaconic acid

[0158] The Salmonella typhimurium SL1344-ΔIct and wild-type SL1344 were cultured in M9 medium and 20 mM glucose as carbon source, and 0, 1, 5, 10, and 20 mM itaconic acid were added. The culture was shaken at 37 °C for 18 h, and then the OD was measured. 600 , the results are as follows Figure 6 As shown in B, compared with the wild-type strain, OD 600 It was significantly reduced, indicating that the sensitivity of the deletion strain to itaconic acid increased significantly after the deletion of the Ict gene.

[0159] Example 4: Virulence evaluation of attenuated Salmonella typhimurium vaccine candidate strain SL1344-ΔIct

[0160] LD of attenuated Salmonella typhimurium vaccine candidate strain SL1344-ΔIct 50 Determination

[0161] Bacterial culture: Bacteria were cultured in LB liquid medium at 37°C overnight. 1 ml of bacterial solution was taken and centrifuged at 5000 rpm for 5 min. The bacteria were washed twice with sterile PBS at 5000 rpm for 5 min. The bacterial concentration was adjusted to 10 9 CFU / ml, and then diluted 10 times.

[0162] The wild-type strain and the deletion strain were injected into 6-week-old BALB / c mice by gavage at different concentrations. The inoculation volume for each mouse was 100 μL, and there were 6 mice in each group. The survival of the mice was observed for 2 consecutive weeks. The results showed that ( Figure 7 Compared with the wild-type SL1344, the virulence of the SL1344-ΔIct deletion strain decreased by about 10 7 times.

[0163] Table 18 The lethality of wild-type SL1344 and mutant strains to 6-week-old BALB / c

[0164]

[0165] Example 5: Evaluation of the immune protection of the attenuated Salmonella typhimurium vaccine candidate strain SL1344-ΔIct

[0166] according to Figure 8 The 6-week-old BALB / c mice were randomly divided into 2 groups, with 6 mice in each group. The SL1344-ΔIct deletion strain was inoculated into 6-week-old BALB / c mice by gavage, and the immunization dose was 1×10 5 CFU / mouse, and a non-immunized PBS control group was set up at the same time. One week after immunization, the mice were reinfected with wild-type SL1344 by gavage injection at a dose of 1×10 4 CFU / mL. Within 14 days after the challenge, there was no death in mice immunized with the SL1344-ΔIct deletion strain, and the protection rate reached 100% ( Figure 8 B and Table 19).

[0167] Table 19 Immune protection efficacy of the deletion strain SL1344-ΔIct against wild-type virus challenge

[0168]

[0169] Example 6: Evaluation of the colonization ability (safety) of the attenuated Salmonella typhimurium vaccine candidate strain SL1344-ΔIct in mice

[0170] Pick a single colony of the SL1344-ΔIct deletion strain and place it in 5 ml of liquid LB medium. Culture it at 37°C with shaking at 180 rpm for 16 h. Wash the bacteria three times with sterile PBS to adjust the bacterial concentration to 1×10 5 CFU / mL.

[0171] Six-week-old BALB / c mice were randomly divided into five groups, with six mice in each group. The SL1344-ΔIct deletion strain was inoculated into the mice by intragastric injection, with an immunization dose of 1×10 5 CFU / piece.

[0172] The mice in each group were autopsied on days 3, 7, 10, 14, and 21 after inoculation. There were 6 mice in each group. The liver, spleen, ileum, and cecum were removed aseptically. After weighing, 1 mL of sterile PBS was added and thoroughly homogenized. 100 μL of the homogenized stock solution was taken and diluted in multiples. Three appropriate dilutions were selected, and 10 μL of each dilution was taken on a drop plate (BG solid plate). Three replicates were made and placed in a 37°C constant temperature incubator for 12 hours. The bacteria were counted and the bacterial load in the internal organs was calculated. The results showed that the bacteria in the liver could be basically cleared 21 days after immunization ( Figure 9-12 ).

[0173] Example 7: The virulence gene Ict of Salmonella typhimurium SL1344 that degrades itaconic acid is widely present in Salmonella enteritidis, Salmonella typhimurium, Salmonella infantis, Salmonella London, and Salmonella Newport

[0174] Based on the Enterobase database (https: / / enterobase.warwick.ac.uk / ), the present invention selected 100 strains of each of 9 common Salmonella serotypes (including Enteritidis, Typhimurium, Derby, Infantis, London, Newport, Rissen, Indiana and Kentucky) for bioinformatics analysis, as shown in Table 20 for details.

[0175] Table 20 9 common Salmonella serotypes

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182] Through homologous sequence alignment and protein sequence analysis, it was found that the highly conserved virulence gene Ict for itaconic acid degradation is commonly found in the genomes of Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Infantis, Salmonella London, and Salmonella Newport, while this virulence gene Ict is absent in Salmonella Delpey, Salmonella Rissen, Salmonella Indiana, and Salmonella Kentucky ( Fig.13 ). Therefore, the "preparation and application of the itaconic acid virulence gene Ict of Salmonella typhimurium in attenuated vaccine candidate strains" proposed in the present invention can be widely applied to the preparation and application of the itaconic acid virulence gene Ict in attenuated vaccine candidate strains of Salmonella Enteritidis, Salmonella Infantis, Salmonella London, and Salmonella Newport. Among them, the amino acid sequence of the protein encoded by the relevant representative itaconic acid-degrading virulence gene Ict sequence in Salmonella Enteritidis, Salmonella Infantis, Salmonella London, and Salmonella Newport is shown in SEQ ID NO.2-5:

[0183] SEQ ID NO.2:

[0184] >tr|A0A3R0Q937|A0A3R0Q937_SALEN Acetyl-CoA hydrolase / transferasefamily protein OS=Salmonella enteritidis OX=149539GN=DTI44_21645PE=3SV=1MMNVNAAYAEKCVTPDEAVTLITSGSHLSMGMFAAEPPALLNALAKRAKRGEINDLRVYCYETANIAGNTIFRYALSDYIHLYSMFITGIERALIHQGIESSGRKIVNYVPSNFHQATRLLADDIGIDTFIHTVSPMDKYGYFNFGTGNDYSTRIARTAKKLIVEVNQYMPRVHGEGAAIHISEIDAIVENHVPLIELPIRTAVAEDIAISQIIASLVPDGACLQMGVGALPELICNALKEHNDLGVHTEALNPGLVSLIQQGVVTNQRKNIDRGMSVFTFAMGQKDMYDYLNDNPSFFSRPVDYVNDPGIIAQNENVVSINATLQIDLTGACNSEHLLGHQYSASGGQLDFVRGAYASKGGRSVITTRSMAANDTISRIVPRLEGPVTTPRTDTHWIVTEFGSVNLKGLSSTERALRIIELAHPNFRQQLRVDAKKMHLI

[0185] SEQ ID NO.3:

[0186] >tr|A0A5Y3D0Y9|A0A5Y3D0Y9_SALIN 4-hydroxybutyrate--acetyl-CoACoAtransferaseOS=Salmonella infantis OX=595 GN=G4J38_000749 PE=3 SV=1MMNVNAVYAEKCVTPDEAVTLITSGSHLSMGMFAAEPPALLNALAKRAKRGEINDLRVYCYETASIAGNTIFRYELSDYIHLYSMFITGIERALIRQGIESSGRKIVNYVPSNFHQATRLLADDIGIDTFIHTVSPMDKYGYFNFGTGNDYSTRIARTAKKLIVEVNKYMPRVHGEGAAIHISEIDAIVENHVPLIELPIRTAVAEDIAISQIIASLVPDGACLQMGVGALPELICNALKEHNDLGVHTEALNPGLVSLIQQGVVTNQRKNIDRGISVFTFAMGQKDMYDYLNDNPSFFSRPVDYVNDPGIIAQNENVVSINATLQIDLTGACNSEYLLGHQYSASGGQLDFVRGAYASKGGRSIITTRSTAANDTISRIVPRLEGPVTTPRTDTHWIVTEFGSVNLKGLSSTERALSIIELAHPNFRQQLRVDAKKMHLI

[0187] SEQ ID NO.4:

[0188] >tr|A0A731UZJ4|A0A731UZJ4_SALET Acetyl-CoAhydrolase / transferasefamily proteinOS=Salmonella enterica subsp.enterica serovar London OX=149390 GN=G4H16_001046PE=3 SV=1

[0189] MMNVNAVYAEKCVTPDEAVTLITSGSHLSMGMFAAEPPALLNALAKRAKRGEINDLR

[0190] VYCYETASIAGNTIFRYELSDYIHLYSMFITGIERALIRQGIESSGRKIVNYVPSNFHQAT

[0191] RLLADDIGIDTFIHTVSPMDKYGYFNFGTGNDYSTRIARTAKKLIVEVNKYMPRVHGE

[0192] GAAIHISEIDAIVENHVPLIELPIRTAVAEDIAISQIIASLVPDGACLQMGVGALPELICNA

[0193] LKEHNDLGVHTEALNPGLVSLIQQGVVTNQRKNIDRGMSVFTFAMGQKDMYDYLN

[0194] DNPSFFSRPVDYVNDPGIIAQNENVVSINATLQIDLTGACNSEYLLGHQYSASGGQLDF

[0195] VRGAYASKGGRSIITTRSTAANDTISRIVPRLEGPVTTPRTDTHWIVTEFDSVNLKGLSS

[0196] TERALRIIELAHPNFRQQLRVDAKKMHLI

[0197] SEQ ID NO.5:

[0198] >tr|A0A3V8SIU3|A0A3V8SIU3_SALNE 4-hydroxybutyrate--acetyl-CoACoAtransferaseOS=Salmonella newport OX=108619 GN=ripAPE=3 SV=1MMNVNAVYAEKCVTPDEAVTLITSGSHLSMGMFAAEPPALLNALAKRAKRGEINDLRVYCYETASIAGNTIFRYELSDYIHLYSMFITGIERALIRQGIESSGRKIVNYVPSNFHQATRLLADDIGIDTFIHTVSPMDKYGYFNFGTGNDYSTRIARTAKKLIVEVNKYMPRVHGEGAAIHISEIDAIVENHVPLIELPIRTAVAEDIAISQIIASLVPDGACLQMGVGALPELICNALKEHNDLGVHTEALNPGLVSLIQQGVVTNQRKNIDRGMSVFTFAMGQKDMYDYLNDNPSFFSRPVDYVNDPGIIAQNENVVSINATLQIDLTGACNSEYLLGHQYSASGGQLDFVRGAYASKGGRSIITTRSTAANDTISRIVPRLEGPVTTPRTDTHWIVTEFGSVNLKGLSSTERALSIIELAHPNFRQQLRVDAKKMHLI。

Claims

1. Use of the Ict gene or its homologous gene with a nucleotide sequence as shown in SEQ ID NO.1 in regulating the virulence of Salmonella, characterized in that: The salmonella includes one or more of salmonella typhimurium, salmonella enteritidis, salmonella infantis, salmonella London or salmonella Newport.

2. Use of the protein encoded by the Ict gene or its homologous gene according to claim 1 in regulating the virulence of Salmonella, characterized in that: The salmonella includes one or more of salmonella typhimurium, salmonella enteritidis, salmonella infantis, salmonella London or salmonella Newport.

3. The use according to claim 1 or 2, characterized in that: The Ict gene is a virulence gene identified from Salmonella typhimurium and has the ability to degrade itaconic acid.

4. Use of the Ict gene or its homologous gene whose nucleotide sequence is shown in SEQ ID NO.1, or a protein encoded by the Ict gene or its homologous gene in regulating the sensitivity of Salmonella to itaconic acid; the Salmonella includes one or more of Salmonella typhimurium, Salmonella enteritidis, Salmonella infantis, Salmonella London or Salmonella Newport.

5. An Ict gene deletion strain, characterized in that: The Ict gene is obtained by knocking out the Ict gene or its homologous gene of the wild strain of Salmonella; the nucleotide sequence of the Ict gene is shown in SEQ ID NO.

1.

6. A method for constructing the Ict gene deletion strain according to claim 4, characterized in that: The following steps are involved: (1) Using Salmonella typhimurium genomic DNA as a template, PCR amplified the upstream and downstream homologous arms of the Ict gene, and the product was homologously recombined with the linear vector and transformed into Escherichia coli to obtain the donor bacteria; (2) The donor bacteria in step (1) are subjected to conjugation transfer and homologous single exchange recombination with wild-type Salmonella typhimurium, and the homologous double exchange is selected using chloramphenicol resistance and sucrose sensitivity to obtain the deletion strain.

7. A method for increasing the sensitivity of Salmonella to itaconic acid, characterized in that: The Ict gene or its homologous gene of Salmonella is knocked out; the nucleotide sequence of the Ict gene is shown in SEQ ID NO.1; the Salmonella includes one or more of Salmonella typhimurium, Salmonella enteritidis, Salmonella infantis, Salmonella London or Salmonella Newport.

8. A method for reducing the virulence of Salmonella, characterized in that: The Ict gene or its homologous gene of Salmonella is knocked out; the nucleotide sequence of the Ict gene is shown in SEQ ID NO.1; the Salmonella includes one or more of Salmonella typhimurium, Salmonella enteritidis, Salmonella infantis, Salmonella London or Salmonella Newport.

9. Use of the Ict gene or its homologous gene whose nucleotide sequence is shown in SEQ ID NO.1, the protein encoded by the Ict gene or its homologous gene, and the deletion strain according to claim 5 in preparing a vaccine to prevent Salmonella; the Salmonella includes one or more of Salmonella typhimurium, Salmonella enteritidis, Salmonella infantis, Salmonella London or Salmonella Newport.

10. A vaccine for preventing Salmonella, characterized in that: It contains the deletion strain according to claim 5; the Salmonella includes one or more of Salmonella typhimurium, Salmonella enteritidis, Salmonella infantis, Salmonella London or Salmonella Newport.

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